In this script, we will perform functional analysis of the identified, differential genes.
In the first part, we will do over-representation analysis (ORA).
In the second part, we will do functional class scoring using gene set enrichement analysis (GSEA).
In the third part we will use SPIA, a tool for Pathway topology.
We will then also shortly try a tool that performs network analysis, namely STRING. String is a Webservice.
Figures generated in this session will be part of the poster. As in the previous section, I would like you to save the plots, and put them in a presentation, together with a short figure legends, and some words describing the figure. # define path to input data here, the input data for this skript is saved
inpDir <- "/proj/courses/transcriptomics/SS23/hw/011_clusterProfiler/inputData"
l_df.res.all <- readRDS(paste(inpDir, "l_df.res.all.rds", sep = "/"))
names(l_df.res.all)
## [1] "s.vs.c" "y.vs.c"
## [3] "s.vs.c.noNa" "y.vs.c.noNa"
## [5] "s.vs.c.noNa.sigPadjUp" "y.vs.c.noNa.sigPadjUp"
## [7] "s.vs.c.noNa.sigPadjDo" "y.vs.c.noNa.sigPadjDo"
## [9] "s.vs.c.noNa.sigPadjFcUp" "y.vs.c.noNa.sigPadjFcUp"
## [11] "s.vs.c.noNa.sigPadjFcDo" "y.vs.c.noNa.sigPadjFcDo"
only y.vs.c.noNa and s.vs.c.noNa are needed (we will subset from full result tables)
df_y.vs.c.noNa <- l_df.res.all$y.vs.c.noNa
df_s.vs.c.noNa <- l_df.res.all$s.vs.c.noNa
dim(df_y.vs.c.noNa)
## [1] 18028 9
dim(df_s.vs.c.noNa)
## [1] 17507 9
head(df_s.vs.c.noNa)
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | ||
|---|---|---|---|---|---|---|
| ENSMUSG00000016494.10 | Cd34 | 8841.095 | -3.881954 | 0.06978926 | 0.000000e+00 | |
| ENSMUSG00000018474.19 | Chd3 | 1595.129 | -5.531728 | 0.10678816 | 0.000000e+00 | |
| ENSMUSG00000004040.17 | Stat3 | 19857.060 | 2.146440 | 0.04342214 | 0.000000e+00 | |
| ENSMUSG00000049804.10 | Armcx4 | 2485.031 | -2.431835 | 0.05679003 | 0.000000e+00 | |
| ENSMUSG00000069045.12 | Ddx3y | 2610.027 | -3.271166 | 0.06843422 | 0.000000e+00 | |
| ENSMUSG00000069049.12 | Eif2s3y | 1312.908 | -3.052586 | 0.08846212 | 6.016023e-262 |
this is the number of sig. reg. genes (padj < 0.05 & abs(log2fc) > 1)
nrow(df_s.vs.c.noNa[df_s.vs.c.noNa$padj < 0.05 &
abs(df_s.vs.c.noNa$log2FoldChange) > 1, ])
## [1] 840
nrow(df_y.vs.c.noNa[df_y.vs.c.noNa$padj < 0.05 &
abs(df_y.vs.c.noNa$log2FoldChange) > 1, ])
## [1] 947
Reactome geneset can be downloaded from here linkToMsigDb
You need to register and login to MsigDb
Download m2.cp.reactome.v2023.1.Mm.symbols.gmt in current working directory.
Now read in and check object
library(GSEABase)
gmt_r <- GSEABase::getGmt(paste(inpDir,
"m2.cp.reactome.v2023.1.Mm.symbols.gmt",
sep = "/"))
gmt_r # GeneSetCollection object
## GeneSetCollection
## names: REACTOME_INTERLEUKIN_6_SIGNALING, REACTOME_APOPTOSIS, ..., REACTOME_ION_CHANNEL_TRANSPORT (1251 total)
## unique identifiers: Stat3, Il6st, ..., Trak1 (9045 total)
## types in collection:
## geneIdType: NullIdentifier (1 total)
## collectionType: NullCollection (1 total)
The next step is to know in which genset each of the genes tested for differential regulation belongs to. Therefore, we generate a table, with two columns, one geneset, and one gene-symbol column.
First convert GeneSetCollection Object into a list.
l_gs <- geneIds(gmt_r)
Then convert it into a data.frame using functions from the tidyverse universe tidyverse
df_gsToGene <- as.data.frame(unnest(enframe(l_gs,
name = "geneset",
value = "symbol"),
cols = "symbol"))
dim(df_gsToGene)
## [1] 70591 2
sum(duplicated(df_gsToGene$symbol))
## [1] 61546
length(unique(df_gsToGene$symbol))
## [1] 9045
head(df_gsToGene, n=4)
geneset <chr> | symbol <chr> | |||
|---|---|---|---|---|
| 1 | REACTOME_INTERLEUKIN_6_SIGNALING | Stat3 | ||
| 2 | REACTOME_INTERLEUKIN_6_SIGNALING | Il6st | ||
| 3 | REACTOME_INTERLEUKIN_6_SIGNALING | Jak2 | ||
| 4 | REACTOME_INTERLEUKIN_6_SIGNALING | Il6 |
The genes in the Reactome database are our “universe”, within we try to find an association between the gensets and our differentially expressed genes. Subset results of differentially expressed genes to genes present in Reactome database.
v_gInR <- unique(df_gsToGene$symbol)
Before subsetting, we check if we have duplicated gene symbols in our result tables
sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 5
Show rows with duplicated gene symbols
df_y.vs.c.noNa[duplicated(df_y.vs.c.noNa$symbol) |
duplicated(df_y.vs.c.noNa$symbol, fromLast=T), ]
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | ||
|---|---|---|---|---|---|---|
| ENSMUSG00000030695.17 | Aldoa | 47325.462764 | 0.35919493 | 0.04578037 | 2.610032e-15 | |
| ENSMUSG00000006378.16 | Gcat | 180.202772 | 0.45479538 | 0.14134739 | 5.070906e-04 | |
| ENSMUSG00000022684.16 | Bfar | 1389.389277 | -0.17867874 | 0.05604261 | 1.216667e-03 | |
| ENSMUSG00000079737.6 | Bfar | 252.347122 | -0.37990952 | 0.13322635 | 2.119518e-03 | |
| ENSMUSG00000117869.2 | Snhg4 | 1305.512128 | 0.13192378 | 0.08222394 | 1.013645e-01 | |
| ENSMUSG00000107877.3 | 4933427D14Rik | 71.787038 | 0.24173733 | 0.20429552 | 1.581147e-01 | |
| ENSMUSG00000114515.3 | Aldoa | 31.777779 | 0.19812268 | 0.26500899 | 3.019270e-01 | |
| ENSMUSG00000020807.16 | 4933427D14Rik | 9.071639 | -0.08267023 | 0.32453327 | 6.006234e-01 | |
| ENSMUSG00000116378.2 | Gcat | 3.794164 | 0.06559034 | 0.34389021 | 6.058058e-01 | |
| ENSMUSG00000117694.2 | Snhg4 | 11.224325 | 0.06758608 | 0.31173056 | 6.873047e-01 |
We see that there are some genes with multiple Ensemble Gene Ids.
We simply remove one of the duplicates randomly, to avoid problems later, with subsetting, merging, …
df_y.vs.c.noNa <- df_y.vs.c.noNa[!duplicated(df_y.vs.c.noNa$symbol), ]
df_s.vs.c.noNa <- df_s.vs.c.noNa[!duplicated(df_s.vs.c.noNa$symbol), ]
sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 0
sum(duplicated(df_s.vs.c.noNa$symbol))
## [1] 0
Next, we subset the data.
df_y.vs.c.noNa <- df_y.vs.c.noNa[df_y.vs.c.noNa$symbol %in% v_gInR, ]
df_s.vs.c.noNa <- df_s.vs.c.noNa[df_s.vs.c.noNa$symbol %in% v_gInR, ]
dim(df_y.vs.c.noNa)
## [1] 6171 9
dim(df_s.vs.c.noNa)
## [1] 6107 9
length(v_gInR)
## [1] 9045
Not all of the 9045 genes in the Reactome Database were expressed in our data. We end up with ~ 6150 genes, that were expressed, and that are in the Reactome database.
These symbols belong to HEMOSTASIS pathway
v_gHemo <- unique(df_gsToGene[grep("HEMOSTASIS", df_gsToGene$geneset), ]$symbol)
length(v_gHemo)
## [1] 628
in diff. exp. table, indicate which genes belong to HEMOSTAIS (==1), and which not (==0)
df_y.vs.c.noNa$isHemostasis <- ifelse(df_y.vs.c.noNa$symbol %in% v_gHemo, 1, 0)
table(df_y.vs.c.noNa$isHemostasis, useNA = "always")
##
## 0 1 <NA>
## 5768 403 0
We see that from the 628 genes of the Hemostasis genset, only 403 are in the Diff.Exp.Table
do it also with s.vs.c diff.exp.table
df_s.vs.c.noNa$isHemostasis <- ifelse(df_s.vs.c.noNa$symbol %in% v_gHemo, 1, 0)
table(df_s.vs.c.noNa$isHemostasis, useNA = "always")
##
## 0 1 <NA>
## 5712 395 0
df_y.vs.c.noNa$isSig <- ifelse(df_y.vs.c.noNa$padj < 0.05 &
abs(df_y.vs.c.noNa$log2FoldChange) > 1, 1, 0)
table(df_y.vs.c.noNa$isSig, useNA = "always")
##
## 0 1 <NA>
## 5758 413 0
df_s.vs.c.noNa$isSig <- ifelse(df_s.vs.c.noNa$padj < 0.05 &
abs(df_s.vs.c.noNa$log2FoldChange) > 1, 1, 0)
table(df_s.vs.c.noNa$isSig, useNA = "always")
##
## 0 1 <NA>
## 5743 364 0
Note that because of subsetting, we only test a fraction of originally identified, sig. genes, for enrichtment in the pathways.
Do it just for Y640F vs. dsRed.
taby <- table(df_y.vs.c.noNa$isSig,
df_y.vs.c.noNa$isHemostasis)
taby_sum <- addmargins(taby, FUN = sum)
## Margins computed over dimensions
## in the following order:
## 1:
## 2:
taby_sum
##
## 0 1 sum
## 0 5408 350 5758
## 1 360 53 413
## sum 5768 403 6171
do it for STAT3
tabs <- table(df_s.vs.c.noNa$isSig,
df_s.vs.c.noNa$isHemostasis)
tabs_sum <- addmargins(tabs, FUN = sum)
## Margins computed over dimensions
## in the following order:
## 1:
## 2:
tabs_sum
##
## 0 1 sum
## 0 5385 358 5743
## 1 327 37 364
## sum 5712 395 6107
In the first row we have the not sig. genes, in the second row the sig. genes, and in the third row we have the sum. In the first column we have the genes that are not in the genset, in the second column we have the genes that are in the genset, and in the third column we have the sum.
Just use Y640F vs. dsRed
fisher.test(taby)
##
## Fisher's Exact Test for Count Data
##
## data: taby
## p-value = 1.344e-06
## alternative hypothesis: true odds ratio is not equal to 1
## 95 percent confidence interval:
## 1.638457 3.109312
## sample estimates:
## odds ratio
## 2.274229
We have a sig. enrichment of homeostasis genes in sig. genes. This is shown by the p-value, but you also have to interpret the odds ratio correctly, to judge if there is an enrichment, or a depletion, of homeostasis genes in the sig. genes. do it also for STAT3
fisher.test(tabs)
##
## Fisher's Exact Test for Count Data
##
## data: tabs
## p-value = 0.00564
## alternative hypothesis: true odds ratio is not equal to 1
## 95 percent confidence interval:
## 1.15771 2.44158
## sample estimates:
## odds ratio
## 1.701804
The odds is the ratio of nr. event occurs / nr. event does not occur.
Odds of homeostasis genes in sig. genes: nr. homeostasis genes in sig. genes / nr. not homoestasis genes in sig. genes.
Odds of homeostasis genes in not sig. genes: nr. homeostasis genes in not sig. genes / nr. not homoestasis genes in not sig. genes.
odds_sigG <- 53/360
odds_notSigG <- 350/5408
odds_sigG
## [1] 0.1472222
odds_notSigG
## [1] 0.06471893
higher proportion of homeostasis genes in sig. genes than in not sig. genes.
Now calculate Odds ratio
odds_ratio <- odds_sigG/odds_notSigG
odds_ratio
## [1] 2.274794
Use df_y.vs.c.noNa already subsetted to correct universe, to see if we can reproduce our finding.
idy <- bitr(df_y.vs.c.noNa$symbol,
fromType = "SYMBOL",
toType = c("ENTREZID"),
OrgDb = "org.Mm.eg.db")
dim(df_y.vs.c.noNa)
## [1] 6171 11
dim(idy)
## [1] 6153 2
head(idy)
SYMBOL <chr> | ENTREZID <chr> | |||
|---|---|---|---|---|
| 1 | Chd3 | 216848 | ||
| 2 | Pgrmc1 | 53328 | ||
| 3 | Stat3 | 20848 | ||
| 4 | Itga9 | 104099 | ||
| 5 | Sgpl1 | 20397 | ||
| 6 | Muc13 | 17063 |
Some symbols did not match an Entrezid, and were dropped.
Check if NA or duplicated Values.
for(i in seq_along(idy)){
print(sum(duplicated(idy[, i])))
print(sum(is.na(idy[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
No.
Use df_s.vs.c.noNa already subsetted to correct universe, to see if we can reproduce our finding.
ids <- bitr(df_s.vs.c.noNa$symbol,
fromType = "SYMBOL",
toType = c("ENTREZID"),
OrgDb = "org.Mm.eg.db")
dim(df_s.vs.c.noNa)
## [1] 6107 11
dim(ids)
## [1] 6090 2
head(ids)
SYMBOL <chr> | ENTREZID <chr> | |||
|---|---|---|---|---|
| 1 | Cd34 | 12490 | ||
| 2 | Chd3 | 216848 | ||
| 3 | Stat3 | 20848 | ||
| 4 | Far2 | 330450 | ||
| 5 | Mid1ip1 | 68041 | ||
| 6 | Uty | 22290 |
for(i in seq_along(ids)){
print(sum(duplicated(ids[, i])))
print(sum(is.na(ids[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
### Merge diff.Exp.Table with idy
```r
df_y.vs.c.noNa.m <- merge(x=df_y.vs.c.noNa,
y=ids,
by.x="symbol",
by.y="SYMBOL",
all=F) # only keep symbols present in both tables
dim(df_y.vs.c.noNa.m)
## [1] 6090 12
sum(is.na(df_y.vs.c.noNa.m$ENTREZID))
## [1] 0
sum(duplicated(df_y.vs.c.noNa.m$ENTREZID))
## [1] 0
head(df_y.vs.c.noNa.m)
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | padj <dbl> | stat <dbl> | ||
|---|---|---|---|---|---|---|---|---|
| 1 | 1600012H06Rik | 475.910354 | 0.06087878 | 0.08499565 | 0.461863686 | 0.630311453 | 0.7357815 | |
| 2 | 2310033P09Rik | 689.922021 | 0.09016197 | 0.08240495 | 0.261587686 | 0.421686026 | 1.1226464 | |
| 3 | 2610008E11Rik | 285.933198 | -0.42819237 | 0.14751424 | 0.001569404 | 0.004987662 | -3.1615342 | |
| 4 | 2810021J22Rik | 23.633663 | -0.31045558 | 0.30298397 | 0.134099749 | 0.250653216 | -1.4981290 | |
| 5 | 9130019O22Rik | 4.386787 | 0.06591355 | 0.33981616 | 0.624836223 | 0.768454100 | 0.4890077 | |
| 6 | A430033K04Rik | 376.791930 | -0.01356353 | 0.09352564 | 0.881051195 | 0.933101263 | -0.1496368 |
#do it for stat3
df_s.vs.c.noNa.m <- merge(x=df_s.vs.c.noNa,
y=ids,
by.x="symbol",
by.y="SYMBOL",
all=F) # only keep symbols present in both tables
dim(df_s.vs.c.noNa.m)
## [1] 6090 12
sum(is.na(df_s.vs.c.noNa.m$ENTREZID))
## [1] 0
sum(duplicated(df_s.vs.c.noNa.m$ENTREZID))
## [1] 0
head(df_s.vs.c.noNa.m)
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | padj <dbl> | ||
|---|---|---|---|---|---|---|---|
| 1 | 1600012H06Rik | 475.910354 | 0.216266728 | 0.08514182 | 8.381174e-03 | 2.659101e-02 | |
| 2 | 2310033P09Rik | 689.922021 | 0.127820993 | 0.08223903 | 1.081567e-01 | 2.309348e-01 | |
| 3 | 2610008E11Rik | 285.933198 | -0.693956278 | 0.15269655 | 9.517084e-07 | 6.601252e-06 | |
| 4 | 2810021J22Rik | 23.633663 | -0.750012423 | 0.39465738 | 5.723083e-03 | 1.914291e-02 | |
| 5 | 9130019O22Rik | 4.386787 | -0.009021925 | 0.30590911 | 9.313940e-01 | 9.648470e-01 | |
| 6 | A430033K04Rik | 376.791930 | -0.234416806 | 0.09649609 | 1.071650e-02 | 3.307717e-02 |
Looks Good, we lost some genes, but have unique Entrezid, without NA values.
# extract sig. res
df_y.vs.c.noNa.sig <- df_y.vs.c.noNa.m[df_y.vs.c.noNa.m$padj < 0.05 &
abs(df_y.vs.c.noNa.m$log2FoldChange) > 1, ]
# dim(df_y.vs.c.noNa.sig)
v_y.vs.c.sig <- as.character(df_y.vs.c.noNa.sig$ENTREZID)
length(v_y.vs.c.sig)
## [1] 413
# do for up reg. Y, extract sig. res
#do it for STAT3
# extract sig. res
df_s.vs.c.noNa.sig <- df_s.vs.c.noNa.m[df_s.vs.c.noNa.m$padj < 0.05 &
abs(df_s.vs.c.noNa.m$log2FoldChange) > 1, ]
# dim(df_y.vs.c.noNa.sig)
v_s.vs.c.sig <- as.character(df_s.vs.c.noNa.sig$ENTREZID)
length(v_s.vs.c.sig)
## [1] 363
# Create background dataset for hypergeometric testing using all genes tested for significance in the results
v_y.vs.c.all <- as.character(df_y.vs.c.noNa.m$ENTREZID)
length(v_y.vs.c.all)
## [1] 6090
#for STAT3
v_s.vs.c.all <- as.character(df_s.vs.c.noNa.m$ENTREZID)
length(v_s.vs.c.all)
## [1] 6090
r_res <- enrichPathway(gene=v_y.vs.c.sig,
pvalueCutoff = 0.05,
readable=TRUE, # convert EntrezId to Gene symbols in result table
organism = "mouse",
universe = v_y.vs.c.all)
class(r_res) # enrichResult
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
#for Stat 3
r_res_s <- enrichPathway(gene=v_s.vs.c.sig,
pvalueCutoff = 0.05,
readable=TRUE, # convert EntrezId to Gene symbols in result table
organism = "mouse",
universe = v_s.vs.c.all)
class(r_res_s) # enrichResult
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
df_res <- r_res@result
dim(df_res)
## [1] 603 9
#stat3
df_res_s <- r_res_s@result
dim(df_res_s)
## [1] 616 9
dim(df_res[df_res$p.adjust < 0.05, ])
## [1] 13 9
#stat3
dim(df_res_s[df_res_s$p.adjust < 0.05, ])
## [1] 20 9
df_res <- data.frame(r_res) # dataframe: only get results with sig padj < 0.05
dim(df_res)
## [1] 13 9
#STAT3
df_res_s <- data.frame(r_res_s) # dataframe: only get results with sig padj < 0.05
dim(df_res_s)
## [1] 20 9
colnames(df_res)
## [1] "ID" "Description" "GeneRatio" "BgRatio" "pvalue"
## [6] "p.adjust" "qvalue" "geneID" "Count"
df_res
ID <chr> | Description <chr> | GeneRatio <chr> | ||
|---|---|---|---|---|
| R-MMU-109582 | R-MMU-109582 | Hemostasis | 50/389 | |
| R-MMU-76002 | R-MMU-76002 | Platelet activation, signaling and aggregation | 30/389 | |
| R-MMU-114608 | R-MMU-114608 | Platelet degranulation | 18/389 | |
| R-MMU-76005 | R-MMU-76005 | Response to elevated platelet cytosolic Ca2+ | 18/389 | |
| R-MMU-500792 | R-MMU-500792 | GPCR ligand binding | 21/389 | |
| R-MMU-372790 | R-MMU-372790 | Signaling by GPCR | 35/389 | |
| R-MMU-6803157 | R-MMU-6803157 | Antimicrobial peptides | 7/389 | |
| R-MMU-388396 | R-MMU-388396 | GPCR downstream signalling | 33/389 | |
| R-MMU-3000178 | R-MMU-3000178 | ECM proteoglycans | 6/389 | |
| R-MMU-375276 | R-MMU-375276 | Peptide ligand-binding receptors | 12/389 |
#STAT3
colnames(df_res_s)
## [1] "ID" "Description" "GeneRatio" "BgRatio" "pvalue"
## [6] "p.adjust" "qvalue" "geneID" "Count"
df_res_s
ID <chr> | ||
|---|---|---|
| R-MMU-500792 | R-MMU-500792 | |
| R-MMU-1296071 | R-MMU-1296071 | |
| R-MMU-372790 | R-MMU-372790 | |
| R-MMU-198933 | R-MMU-198933 | |
| R-MMU-388396 | R-MMU-388396 | |
| R-MMU-112316 | R-MMU-112316 | |
| R-MMU-1266738 | R-MMU-1266738 | |
| R-MMU-977068 | R-MMU-977068 | |
| R-MMU-373080 | R-MMU-373080 | |
| R-MMU-373076 | R-MMU-373076 |
ClusterProfiler does not give you an odds ratio, to judge if geneset is enriched, or depleted. But you can calculate it by deviding GeneRation / BgRatio.
map_dbl(df_res$GeneRatio, ~ eval(parse(text=.x)))/map_dbl(df_res$BgRatio, ~ eval(parse(text=.x)))
## [1] 2.119548 2.494002 3.409521 3.284783 2.618702 2.006668 6.161651 2.007367
## [9] 6.906466 3.388078 2.602437 2.992802 2.418426
#STAT3
map_dbl(df_res_s$GeneRatio, ~ eval(parse(text=.x)))/map_dbl(df_res_s$BgRatio, ~ eval(parse(text=.x)))
## [1] 3.243290 6.508274 2.334002 4.489381 2.201172 2.664805 2.076731 7.691596
## [9] 5.150025 2.758942 6.508274 2.563865 2.784553 2.079739 2.079739 2.603309
## [17] 2.682679 4.230378 2.354297 1.677770
These were our results when we calculated it manually:
0 1 sum
0 5408 350 5758
1 360 53 413
sum 5768 403 6171
Fisher's Exact Test for Count Data
data: tab
p-value = 1e-06
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
1.6 3.1
sample estimates:
odds ratio
2.3
The results are a bit different. For manual calculation, we used gene symbols, ClusterProfiler used EntrezIds. Furthermore, we possibly used different versions of Reactome Database.
To show fold change in Reactome Pathway illustration, a vector of log2fc of sig. genes, with genes named by Entrezid, is needed.
v_nam <- df_y.vs.c.noNa.sig$log2FoldChange
names(v_nam) <- df_y.vs.c.noNa.sig$ENTREZID
v_nam[1:4]
## 56325 76491 66082 11363
## 1.792428 1.067964 -1.808291 -5.136837
#STAT3
v_nam_s <- df_s.vs.c.noNa.sig$log2FoldChange
names(v_nam_s) <- df_s.vs.c.noNa.sig$ENTREZID
v_nam_s[1:4]
## 56325 66082 11363 264895
## 1.862131 -3.692707 -3.571212 -2.117135
Now plot Reactome pathway Hemostasis.
viewPathway("Hemostasis",
readable = T,
foldChange = v_nam,
organism = "mouse")
#STAT3
viewPathway("Hemostasis",
readable = T,
foldChange = v_nam_s,
organism = "mouse")
Genes up or down-regulated are indicated. Brown circles indicate genes not within sig. genes. Gray lines indicates known interactions.
Here is how the pathway looks in Reactome pathway browser pathwayBrowser.
Note that some of our enriched Pathways are in the hirarchy below Hemostasis. So probably there are similar genes in some of the pathways. Plot heatmap.
heatplot(r_res, foldChange=v_nam, showCategory=12) +
theme(axis.text.x = element_text(size = 4))
#STAT3
heatplot(r_res_s, foldChange=v_nam, showCategory=12) +
theme(axis.text.x = element_text(size = 4))
also plot classical Reactome pathway, and check how it looks with pathview
viewPathway("Platelet calcium homeostasis",
readable = T,
foldChange = v_nam,
organism = "mouse")
#STAT3
viewPathway("Platelet calcium homeostasis",
readable = T,
foldChange = v_nam_s,
organism = "mouse")
This is how it looks in the Reactome Pathway browser link
reprocess diff.exp.tables (now they are subsetted to genes in Reactome Pathway)
df_y.vs.c.noNa <- l_df.res.all$y.vs.c.noNa
df_s.vs.c.noNa <- l_df.res.all$s.vs.c.noNa
dim(df_y.vs.c.noNa)
## [1] 18028 9
dim(df_s.vs.c.noNa)
## [1] 17507 9
remove duplicated gene symbols, if there
sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 5
df_y.vs.c.noNa <- df_y.vs.c.noNa[!duplicated(df_y.vs.c.noNa$symbol), ]
sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 0
cluster profiles needs Enrezid -> convert symbols
#
idy.y <- bitr(df_y.vs.c.noNa$symbol,
fromType = "SYMBOL",
toType = c("ENTREZID"),
OrgDb = "org.Mm.eg.db")
head(idy.y)
SYMBOL <chr> | ENTREZID <chr> | |||
|---|---|---|---|---|
| 1 | Chd3 | 216848 | ||
| 2 | Pgrmc1 | 53328 | ||
| 3 | Stat3 | 20848 | ||
| 4 | Itga9 | 104099 | ||
| 5 | Sgpl1 | 20397 | ||
| 6 | Muc13 | 17063 |
for(i in seq_along(idy.y)){
print(sum(duplicated(idy.y[, i])))
print(sum(is.na(idy.y[, i])))
}
## [1] 1
## [1] 0
## [1] 0
## [1] 0
remove duplicated symbol
idy.y <- idy.y[!duplicated(idy.y$SYMBOL), ]
for(i in seq_along(idy.y)){
print(sum(duplicated(idy.y[, i])))
print(sum(is.na(idy.y[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
ids.s <- bitr(df_s.vs.c.noNa$symbol,
fromType = "SYMBOL",
toType = c("ENTREZID"),
OrgDb = "org.Mm.eg.db")
head(ids.s)
SYMBOL <chr> | ENTREZID <chr> | |||
|---|---|---|---|---|
| 1 | Cd34 | 12490 | ||
| 2 | Chd3 | 216848 | ||
| 3 | Stat3 | 20848 | ||
| 4 | Armcx4 | 100503043 | ||
| 5 | Ddx3y | 26900 | ||
| 6 | Eif2s3y | 26908 |
for(i in seq_along(ids.s)){
print(sum(duplicated(ids.s[, i])))
print(sum(is.na(ids.s[, i])))
}
## [1] 1
## [1] 0
## [1] 0
## [1] 0
remove duplicated symbol
ids.s <- ids.s[!duplicated(ids.s$SYMBOL), ]
check if all duplicated values removed
#
for(i in seq_along(idy.y)){
print(sum(duplicated(idy.y[, i])))
print(sum(is.na(idy.y[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
dim(idy.y)
## [1] 16235 2
for(i in seq_along(ids.s)){
print(sum(duplicated(ids.s[, i])))
print(sum(is.na(ids.s[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
dim(ids.s)
## [1] 15843 2
merge diff.exp.Table with EntrezToSymbol conversion table
df_y.vs.c.noNa.m <- merge(x=df_y.vs.c.noNa,
y=idy.y,
by.x="symbol",
by.y="SYMBOL",
all=F)
dim(df_y.vs.c.noNa.m)
## [1] 16235 10
head(df_y.vs.c.noNa.m)
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | padj <dbl> | stat <dbl> | ||
|---|---|---|---|---|---|---|---|---|
| 1 | 0610009B22Rik | 232.167355 | 0.26894048 | 0.12056811 | 0.01766358 | 0.04427685 | 2.3725958 | |
| 2 | 0610009E02Rik | 9.487715 | 0.10464918 | 0.31949663 | 0.53740434 | 0.69705197 | 0.6167429 | |
| 3 | 0610009L18Rik | 18.354738 | 0.22098446 | 0.30532700 | 0.26394260 | 0.42432291 | 1.1171210 | |
| 4 | 0610010K14Rik | 15.129557 | -0.02823405 | 0.28513465 | 0.87145474 | 0.92802801 | -0.1618109 | |
| 5 | 0610030E20Rik | 1081.724994 | 0.02836226 | 0.07019858 | 0.68084522 | 0.80799669 | 0.4113100 | |
| 6 | 0610033M10Rik | 1.646061 | 0.04618991 | 0.35345241 | 0.62190168 | 0.76624135 | 0.4931570 |
df_s.vs.c.noNa.m <- merge(x=df_s.vs.c.noNa,
y=ids.s,
by.x="symbol",
by.y="SYMBOL",
all=F)
dim(df_s.vs.c.noNa.m)
## [1] 15848 10
head(df_s.vs.c.noNa.m)
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | padj <dbl> | ||
|---|---|---|---|---|---|---|---|
| 1 | 0610009B22Rik | 232.167355 | 0.111259102 | 0.11813052 | 3.118127e-01 | 5.053138e-01 | |
| 2 | 0610009E02Rik | 9.487715 | -0.099422854 | 0.30303284 | 4.788268e-01 | 6.688599e-01 | |
| 3 | 0610009L18Rik | 18.354738 | 0.046590611 | 0.25753714 | 7.718249e-01 | 8.809140e-01 | |
| 4 | 0610010K14Rik | 15.129557 | 0.001606264 | 0.26557513 | 9.932166e-01 | 9.969180e-01 | |
| 5 | 0610030E20Rik | 1081.724994 | 0.467729701 | 0.07041157 | 1.100300e-11 | 1.363266e-10 | |
| 6 | 0610038B21Rik | 40.326866 | 0.119189081 | 0.23040933 | 4.776907e-01 | 6.677969e-01 |
check for duplicated and NA after merging
#
for(i in c("symbol", "ENTREZID")){
print(sum(duplicated(df_y.vs.c.noNa.m[, i])))
print(sum(is.na(df_y.vs.c.noNa.m[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
for(i in c("symbol", "ENTREZID")){
print(sum(duplicated(df_s.vs.c.noNa.m[, i])))
print(sum(is.na(df_s.vs.c.noNa.m[, i])))
}
## [1] 5
## [1] 0
## [1] 5
## [1] 0
make unique for gene symbol and entrezid
#
df_y.vs.c.noNa.m <- df_y.vs.c.noNa.m[!duplicated(df_y.vs.c.noNa.m$ENTREZID) &
!is.na(df_y.vs.c.noNa.m$ENTREZID), ]
df_s.vs.c.noNa.m <- df_s.vs.c.noNa.m[!duplicated(df_s.vs.c.noNa.m$ENTREZID) &
!is.na(df_s.vs.c.noNa.m$ENTREZID), ]
evaluate that removing of duplicates and NA values worked
for(i in c("symbol", "ENTREZID")){
print(sum(duplicated(df_y.vs.c.noNa.m[, i])))
print(sum(is.na(df_y.vs.c.noNa.m[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
for(i in c("symbol", "ENTREZID")){
print(sum(duplicated(df_s.vs.c.noNa.m[, i])))
print(sum(is.na(df_s.vs.c.noNa.m[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
extract sig. res, and make character vector with Entrezid as names
# extract sig. res
df_y.vs.c.noNa.sig <- df_y.vs.c.noNa.m[df_y.vs.c.noNa.m$padj < 0.05 &
abs(df_y.vs.c.noNa.m$log2FoldChange) > 1, ]
dim(df_y.vs.c.noNa.sig)
## [1] 898 10
v_y.vs.c.sig <- unique(as.character(df_y.vs.c.noNa.sig$ENTREZID))
length(v_y.vs.c.sig)
## [1] 898
sum(duplicated(names(v_y.vs.c.sig)))
## [1] 0
df_s.vs.c.noNa.sig <- df_s.vs.c.noNa.m[df_s.vs.c.noNa.m$padj < 0.05 &
abs(df_s.vs.c.noNa.m$log2FoldChange) > 1, ]
dim(df_s.vs.c.noNa.sig)
## [1] 802 10
v_s.vs.c.sig <- as.character(df_s.vs.c.noNa.sig$ENTREZID)
length(v_s.vs.c.sig)
## [1] 802
sum(duplicated(names(v_s.vs.c.sig)))
## [1] 0
Create background dataset for hypergeometric testing using all genes tested for significance in the results
#
v_y.vs.c.all <- as.character(df_y.vs.c.noNa.m$ENTREZID)
v_s.vs.c.all <- as.character(df_s.vs.c.noNa.m$ENTREZID)
length(v_y.vs.c.all)
## [1] 16235
length(v_s.vs.c.all)
## [1] 15843
create common background for s.vs.c and y.vs.c, to compare enriched Kegg Pathways of these comparisions (s. below)
#
v_y.vs.c.all <- unique(c(v_s.vs.c.all, v_y.vs.c.all))
length(v_y.vs.c.all)
## [1] 16235
run Kegg (for now only on Y640F vs. dsRed)
kky <- enrichKEGG(gene = v_y.vs.c.sig,
organism = 'mmu',
pvalueCutoff = 0.05,
universe = v_y.vs.c.all)
kky <- setReadable(kky, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
class(kky)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
#STAT3
kks <- enrichKEGG(gene = v_s.vs.c.sig,
organism = 'mmu',
pvalueCutoff = 0.05,
universe = v_s.vs.c.all)
kks <- setReadable(kks, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
class(kks)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
extract result table, and check which pathways are enriched
df_kky <- kky@result
df_kky
ID <chr> | ||
|---|---|---|
| mmu04514 | mmu04514 | |
| mmu04630 | mmu04630 | |
| mmu04080 | mmu04080 | |
| mmu04640 | mmu04640 | |
| mmu04020 | mmu04020 | |
| mmu00982 | mmu00982 | |
| mmu04610 | mmu04610 | |
| mmu00980 | mmu00980 | |
| mmu05320 | mmu05320 | |
| mmu05200 | mmu05200 |
#STAT3
df_kks <- kks@result
df_kks
ID <chr> | ||
|---|---|---|
| mmu04640 | mmu04640 | |
| mmu04060 | mmu04060 | |
| mmu04514 | mmu04514 | |
| mmu05204 | mmu05204 | |
| mmu00982 | mmu00982 | |
| mmu00980 | mmu00980 | |
| mmu05320 | mmu05320 | |
| mmu05330 | mmu05330 | |
| mmu05150 | mmu05150 | |
| mmu04061 | mmu04061 |
subset to pathways with sig. p.adjust
# dim(data.frame(kk))
# max(data.frame(kk)$p.adjust)
# data.frame(enrichResult) gives you the sig. gs. with p.adjust < 0.05
df_kky.sig <- df_kky[df_kky$p.adjust < 0.05, ]
as.matrix(df_kky.sig$Description)
## [,1]
## [1,] "Cell adhesion molecules - Mus musculus (house mouse)"
## [2,] "JAK-STAT signaling pathway - Mus musculus (house mouse)"
## [3,] "Neuroactive ligand-receptor interaction - Mus musculus (house mouse)"
## [4,] "Hematopoietic cell lineage - Mus musculus (house mouse)"
## [5,] "Calcium signaling pathway - Mus musculus (house mouse)"
## [6,] "Drug metabolism - cytochrome P450 - Mus musculus (house mouse)"
## [7,] "Complement and coagulation cascades - Mus musculus (house mouse)"
## [8,] "Metabolism of xenobiotics by cytochrome P450 - Mus musculus (house mouse)"
## [9,] "Autoimmune thyroid disease - Mus musculus (house mouse)"
## [10,] "Pathways in cancer - Mus musculus (house mouse)"
## [11,] "Allograft rejection - Mus musculus (house mouse)"
## [12,] "Cytokine-cytokine receptor interaction - Mus musculus (house mouse)"
## [13,] "Graft-versus-host disease - Mus musculus (house mouse)"
## [14,] "Transcriptional misregulation in cancer - Mus musculus (house mouse)"
## [15,] "PI3K-Akt signaling pathway - Mus musculus (house mouse)"
## [16,] "Intestinal immune network for IgA production - Mus musculus (house mouse)"
## [17,] "Chemical carcinogenesis - DNA adducts - Mus musculus (house mouse)"
## [18,] "Asthma - Mus musculus (house mouse)"
#STAT3
df_kks.sig <- df_kks[df_kks$p.adjust < 0.05, ]
as.matrix(df_kks.sig$Description)
## [,1]
## [1,] "Hematopoietic cell lineage - Mus musculus (house mouse)"
## [2,] "Cytokine-cytokine receptor interaction - Mus musculus (house mouse)"
## [3,] "Cell adhesion molecules - Mus musculus (house mouse)"
## [4,] "Chemical carcinogenesis - DNA adducts - Mus musculus (house mouse)"
## [5,] "Drug metabolism - cytochrome P450 - Mus musculus (house mouse)"
## [6,] "Metabolism of xenobiotics by cytochrome P450 - Mus musculus (house mouse)"
## [7,] "Autoimmune thyroid disease - Mus musculus (house mouse)"
## [8,] "Allograft rejection - Mus musculus (house mouse)"
## [9,] "Staphylococcus aureus infection - Mus musculus (house mouse)"
## [10,] "Viral protein interaction with cytokine and cytokine receptor - Mus musculus (house mouse)"
## [11,] "Phagosome - Mus musculus (house mouse)"
## [12,] "Leishmaniasis - Mus musculus (house mouse)"
## [13,] "Inflammatory bowel disease - Mus musculus (house mouse)"
## [14,] "Pathways in cancer - Mus musculus (house mouse)"
## [15,] "Graft-versus-host disease - Mus musculus (house mouse)"
## [16,] "Type I diabetes mellitus - Mus musculus (house mouse)"
## [17,] "Viral myocarditis - Mus musculus (house mouse)"
## [18,] "Intestinal immune network for IgA production - Mus musculus (house mouse)"
## [19,] "Fluid shear stress and atherosclerosis - Mus musculus (house mouse)"
## [20,] "Steroid hormone biosynthesis - Mus musculus (house mouse)"
## [21,] "Asthma - Mus musculus (house mouse)"
## [22,] "JAK-STAT signaling pathway - Mus musculus (house mouse)"
## [23,] "Complement and coagulation cascades - Mus musculus (house mouse)"
## [24,] "Rheumatoid arthritis - Mus musculus (house mouse)"
## [25,] "Antigen processing and presentation - Mus musculus (house mouse)"
## [26,] "Tuberculosis - Mus musculus (house mouse)"
## [27,] "Axon guidance - Mus musculus (house mouse)"
## [28,] "Ascorbate and aldarate metabolism - Mus musculus (house mouse)"
## [29,] "Amoebiasis - Mus musculus (house mouse)"
## [30,] "Th17 cell differentiation - Mus musculus (house mouse)"
## [31,] "Bile secretion - Mus musculus (house mouse)"
get the similarity matrix between terms by calculating Jaccard similarity coefficient (used by some plots later for clustering of genesets).
kk2y <- pairwise_termsim(kky)
#STAT3
kk2s <- pairwise_termsim(kks)
In general, plots are ggplots, and can be modified using ggplot functions
Barplot
barplot(kky, showCategory = 10)
#STAT3
barplot(kks, showCategory = 10)
dotplot
dotplot(kky, showCategory=12) +
ggtitle("dotplot for ORA") +
theme(axis.text.y = element_text(size = 7))
#STAT3
dotplot(kks, showCategory=12) +
ggtitle("dotplot for ORA") +
theme(axis.text.y = element_text(size = 7))
df_kky.sig[df_kky.sig$Description %in% "Allograft rejection - Mus musculus (house mouse)", ]
ID <chr> | Description <chr> | GeneRatio <chr> | BgRatio <chr> | ||
|---|---|---|---|---|---|
| mmu05330 | mmu05330 | Allograft rejection - Mus musculus (house mouse) | 7/370 | 22/5184 |
#STAT3
df_kks.sig[df_kks.sig$Description %in% "Allograft rejection - Mus musculus (house mouse)", ]
ID <chr> | Description <chr> | GeneRatio <chr> | BgRatio <chr> | ||
|---|---|---|---|---|---|
| mmu05330 | mmu05330 | Allograft rejection - Mus musculus (house mouse) | 8/310 | 21/5125 |
heatmap
heatplot(kky, foldChange=v_nam, showCategory=12) +
theme(axis.text.x = element_text(size = 3.75))
#STAT3
heatplot(kks, foldChange=v_nam_s, showCategory=12) +
theme(axis.text.x = element_text(size = 3.75))
treeplot
treeplot(kk2y, hclust_method = "average")
#STAT3
treeplot(kk2s, hclust_method = "average")
cnetplot
cnetplot(kk2y, categorySize = "pvalue", foldChange = v_nam,
showCategory=5, cex_label_gene=0.5, cex_label_category = 0.5)
#STAT3
cnetplot(kk2s, categorySize = "pvalue", foldChange = v_nam_s,
showCategory=5, cex_label_gene=0.5, cex_label_category = 0.5)
enrichplot
enrichplot::emapplot(kk2y,
# cex_category=1.5,
layout="kk",
showCategory=18,
cex_label_category = 0.5)
#STAT3
enrichplot::emapplot(kk2s,
# cex_category=1.5,
layout="kk",
showCategory=18,
cex_label_category = 0.5)
genesets with common genes cluster together, and are connected with edges. Thickness of edges correlates with proportion of overlapping genes.
Enrichplot can be further manipulated in an interactive way using the Cytoscape Plugin Enrichment Map link. Another Possibility to visualises clusters of similar pathways as an enrichment network is aPEAR link.
You can also access a specific pathway in the the Kegg pathway browser
browseKEGG(kky, 'mmu04630')
#STAT3
browseKEGG(kks, 'mmu04630')
or visualize a pathway, including the fold change of sig. genes in the pathway
mmu04630 <- pathview(gene.data = v_nam,
pathway.id = "mmu04630",
species = "mmu",
limit = list(gene=max(abs(v_nam)), cpd=1))
#STAT
mmu04630_s <- pathview(gene.data = v_nam_s,
pathway.id = "mmu04630",
species = "mmu",
limit = list(gene=max(abs(v_nam_s)), cpd=1))
# png is saved in current directory
mmu04630
load hallmark GMT file from MsigDb
file was downloaded from here msigdb.
gmt_h <- GSEABase::getGmt(paste(inpDir, "mh.all.v2023.1.Mm.entrez.gmt", sep = "/"))
gmt_h # GeneSetCollection object
## GeneSetCollection
## names: HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_HYPOXIA, ..., HALLMARK_PANCREAS_BETA_CELLS (50 total)
## unique identifiers: 23849, 16449, ..., 18088 (4289 total)
## types in collection:
## geneIdType: NullIdentifier (1 total)
## collectionType: NullCollection (1 total)
convert to list of genesets
l_gs <- geneIds(gmt_h)
convert to data.frame in long format
df_gsToGene <- as.data.frame(unnest(enframe(l_gs, name = "geneset", value = "symbol"),
cols = "symbol"))
dim(df_gsToGene)
## [1] 7189 2
head(df_gsToGene)
geneset <chr> | symbol <chr> | |||
|---|---|---|---|---|
| 1 | HALLMARK_TNFA_SIGNALING_VIA_NFKB | 23849 | ||
| 2 | HALLMARK_TNFA_SIGNALING_VIA_NFKB | 16449 | ||
| 3 | HALLMARK_TNFA_SIGNALING_VIA_NFKB | 19347 | ||
| 4 | HALLMARK_TNFA_SIGNALING_VIA_NFKB | 14528 | ||
| 5 | HALLMARK_TNFA_SIGNALING_VIA_NFKB | 14595 | ||
| 6 | HALLMARK_TNFA_SIGNALING_VIA_NFKB | 15894 |
nr of genes in Hallmark
(l_g_hm <- length(unique(df_gsToGene$symbol)))
## [1] 4289
nr of genes in diff.exp.table and hallmark
(l_is_hm.deg <- length(intersect(unique(df_gsToGene$symbol),
v_y.vs.c.all)))
## [1] 3195
#Stat3
(l_is_hm.deg_s <- length(intersect(unique(df_gsToGene$symbol),
v_s.vs.c.all)))
## [1] 3169
ClusterProfiler has function “enricher” for performing over-representation analysis of various gensets. As an input, it needs again the vector of sig. genes, IDs as Entrezid, a dataframe with two columns names “geneset” and “symbol”, and some additional, optional parameters. We also define the background genset, defined by the parameter “universe”, taking all genes that were tested.
hy <- enricher(v_y.vs.c.sig,
TERM2GENE = df_gsToGene,
universe = v_y.vs.c.all)
class(hy)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
data.frame(hy)
ID <chr> | ||
|---|---|---|
| HALLMARK_INTERFERON_GAMMA_RESPONSE | HALLMARK_INTERFERON_GAMMA_RESPONSE | |
| HALLMARK_INFLAMMATORY_RESPONSE | HALLMARK_INFLAMMATORY_RESPONSE | |
| HALLMARK_ANGIOGENESIS | HALLMARK_ANGIOGENESIS | |
| HALLMARK_IL2_STAT5_SIGNALING | HALLMARK_IL2_STAT5_SIGNALING | |
| HALLMARK_HYPOXIA | HALLMARK_HYPOXIA | |
| HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION | HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION | |
| HALLMARK_INTERFERON_ALPHA_RESPONSE | HALLMARK_INTERFERON_ALPHA_RESPONSE | |
| HALLMARK_TNFA_SIGNALING_VIA_NFKB | HALLMARK_TNFA_SIGNALING_VIA_NFKB | |
| HALLMARK_HEME_METABOLISM | HALLMARK_HEME_METABOLISM | |
| HALLMARK_KRAS_SIGNALING_UP | HALLMARK_KRAS_SIGNALING_UP |
#STAT3
hs <- enricher(v_s.vs.c.sig,
TERM2GENE = df_gsToGene,
universe = v_s.vs.c.all)
class(hs)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
data.frame(hs)
ID <chr> | ||
|---|---|---|
| HALLMARK_INTERFERON_GAMMA_RESPONSE | HALLMARK_INTERFERON_GAMMA_RESPONSE | |
| HALLMARK_KRAS_SIGNALING_UP | HALLMARK_KRAS_SIGNALING_UP | |
| HALLMARK_IL6_JAK_STAT3_SIGNALING | HALLMARK_IL6_JAK_STAT3_SIGNALING | |
| HALLMARK_HYPOXIA | HALLMARK_HYPOXIA | |
| HALLMARK_INFLAMMATORY_RESPONSE | HALLMARK_INFLAMMATORY_RESPONSE | |
| HALLMARK_ESTROGEN_RESPONSE_EARLY | HALLMARK_ESTROGEN_RESPONSE_EARLY | |
| HALLMARK_INTERFERON_ALPHA_RESPONSE | HALLMARK_INTERFERON_ALPHA_RESPONSE | |
| HALLMARK_IL2_STAT5_SIGNALING | HALLMARK_IL2_STAT5_SIGNALING |
barplot(hy, categories=12)
#Stat3
barplot(hs, categories=12)
compare Y640F.vs.dsRed with STAT3-wt.vs.dsRed. Put sig. genelists into a list.
l_sigEntrez <- list(y.vs.c = v_y.vs.c.sig,
s.vs.c = v_s.vs.c.sig)
Run function compareCluster
ck <- compareCluster(geneCluster = l_sigEntrez,
fun="enrichKEGG",
organism="mmu", pvalueCutoff=0.05, pAdjustMethod="BH")
ck <- setReadable(ck, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
class(ck)
## [1] "compareClusterResult"
## attr(,"package")
## [1] "DOSE"
extract sig. results
res_comp <- data.frame(ck)
dim(res_comp)
## [1] 55 10
colnames(res_comp)
## [1] "Cluster" "ID" "Description" "GeneRatio" "BgRatio"
## [6] "pvalue" "p.adjust" "qvalue" "geneID" "Count"
head(res_comp, n=10)
Cluster <fct> | ID <chr> | ||
|---|---|---|---|
| 1 | y.vs.c | mmu04630 | |
| 2 | y.vs.c | mmu04640 | |
| 3 | y.vs.c | mmu04151 | |
| 4 | y.vs.c | mmu05202 | |
| 5 | y.vs.c | mmu04514 | |
| 6 | y.vs.c | mmu04015 | |
| 7 | y.vs.c | mmu04014 | |
| 8 | y.vs.c | mmu04668 | |
| 9 | y.vs.c | mmu04658 | |
| 10 | y.vs.c | mmu01521 |
tail(res_comp, n=10)
Cluster <fct> | ID <chr> | Description <chr> | ||
|---|---|---|---|---|
| 46 | s.vs.c | mmu04612 | Antigen processing and presentation - Mus musculus (house mouse) | |
| 47 | s.vs.c | mmu05202 | Transcriptional misregulation in cancer - Mus musculus (house mouse) | |
| 48 | s.vs.c | mmu05145 | Toxoplasmosis - Mus musculus (house mouse) | |
| 49 | s.vs.c | mmu05166 | Human T-cell leukemia virus 1 infection - Mus musculus (house mouse) | |
| 50 | s.vs.c | mmu04151 | PI3K-Akt signaling pathway - Mus musculus (house mouse) | |
| 51 | s.vs.c | mmu05320 | Autoimmune thyroid disease - Mus musculus (house mouse) | |
| 52 | s.vs.c | mmu05169 | Epstein-Barr virus infection - Mus musculus (house mouse) | |
| 53 | s.vs.c | mmu05332 | Graft-versus-host disease - Mus musculus (house mouse) | |
| 54 | s.vs.c | mmu05163 | Human cytomegalovirus infection - Mus musculus (house mouse) | |
| 55 | s.vs.c | mmu04014 | Ras signaling pathway - Mus musculus (house mouse) |
unique(res_comp$Description)
## [1] "JAK-STAT signaling pathway - Mus musculus (house mouse)"
## [2] "Hematopoietic cell lineage - Mus musculus (house mouse)"
## [3] "PI3K-Akt signaling pathway - Mus musculus (house mouse)"
## [4] "Transcriptional misregulation in cancer - Mus musculus (house mouse)"
## [5] "Cell adhesion molecules - Mus musculus (house mouse)"
## [6] "Rap1 signaling pathway - Mus musculus (house mouse)"
## [7] "Ras signaling pathway - Mus musculus (house mouse)"
## [8] "TNF signaling pathway - Mus musculus (house mouse)"
## [9] "Th1 and Th2 cell differentiation - Mus musculus (house mouse)"
## [10] "EGFR tyrosine kinase inhibitor resistance - Mus musculus (house mouse)"
## [11] "Calcium signaling pathway - Mus musculus (house mouse)"
## [12] "Intestinal immune network for IgA production - Mus musculus (house mouse)"
## [13] "Toxoplasmosis - Mus musculus (house mouse)"
## [14] "Acute myeloid leukemia - Mus musculus (house mouse)"
## [15] "Melanoma - Mus musculus (house mouse)"
## [16] "Epstein-Barr virus infection - Mus musculus (house mouse)"
## [17] "Hepatitis C - Mus musculus (house mouse)"
## [18] "Asthma - Mus musculus (house mouse)"
## [19] "Inflammatory bowel disease - Mus musculus (house mouse)"
## [20] "Focal adhesion - Mus musculus (house mouse)"
## [21] "Leishmaniasis - Mus musculus (house mouse)"
## [22] "Cytokine-cytokine receptor interaction - Mus musculus (house mouse)"
## [23] "Phagosome - Mus musculus (house mouse)"
## [24] "Viral protein interaction with cytokine and cytokine receptor - Mus musculus (house mouse)"
## [25] "Tuberculosis - Mus musculus (house mouse)"
## [26] "Fluid shear stress and atherosclerosis - Mus musculus (house mouse)"
## [27] "Th17 cell differentiation - Mus musculus (house mouse)"
## [28] "Rheumatoid arthritis - Mus musculus (house mouse)"
## [29] "Axon guidance - Mus musculus (house mouse)"
## [30] "Allograft rejection - Mus musculus (house mouse)"
## [31] "MAPK signaling pathway - Mus musculus (house mouse)"
## [32] "Kaposi sarcoma-associated herpesvirus infection - Mus musculus (house mouse)"
## [33] "Chemical carcinogenesis - DNA adducts - Mus musculus (house mouse)"
## [34] "Drug metabolism - cytochrome P450 - Mus musculus (house mouse)"
## [35] "Viral myocarditis - Mus musculus (house mouse)"
## [36] "Metabolism of xenobiotics by cytochrome P450 - Mus musculus (house mouse)"
## [37] "Amoebiasis - Mus musculus (house mouse)"
## [38] "Antigen processing and presentation - Mus musculus (house mouse)"
## [39] "Human T-cell leukemia virus 1 infection - Mus musculus (house mouse)"
## [40] "Autoimmune thyroid disease - Mus musculus (house mouse)"
## [41] "Graft-versus-host disease - Mus musculus (house mouse)"
## [42] "Human cytomegalovirus infection - Mus musculus (house mouse)"
get distance matrix for plots
ck2 <- pairwise_termsim(ck)
dotplot
dotplot(ck, showCategory = 40, font.size = 8)
dotplot
dotplot(ck, showCategory=12) +
ggtitle("dotplot for ORA") +
theme(axis.text.y = element_text(size = 7))
heatmap
heatplot(ck)
## Error in (function (classes, fdef, mtable) : unable to find an inherited method for function 'heatplot' for signature '"compareClusterResult"'
treeplot
treeplot(ck2, hclust_method = "average")
cnetplot
cnetplot(ck2, categorySize = "pvalue",
showCategory=5, cex_label_gene=0.5, cex_label_category = 0.5)
you see common and specific enriched pathways; you see, whether in common pathways, common or specific genes are enriched.
enrichplot
enrichplot::emapplot(ck2, cex_category=1.5,layout="kk",
showCategory=20)
We need to create a ranked list from our diff. exp. analysis. We rank the list based on the -log10(p-value). In addition, we indicate the direction of regulation, by multiplying the minus log10 pvalue with the sign of the fold change.
We use already above prepared dataframe, with unique Entrezid, and no NA values. We check for duplicated and NA values, and 0 values (will be inf when log transformed) in pvalue column. We also check log2FC (there, just NA values would be a problem).
for(i in c("pvalue", "log2FoldChange")){
print(sum(duplicated(df_y.vs.c.noNa.m[, i])))
print(sum(is.na(df_y.vs.c.noNa.m[, i])))
print(sum(df_y.vs.c.noNa.m[, i] == 0))
}
## [1] 2
## [1] 0
## [1] 2
## [1] 1
## [1] 0
## [1] 0
#STAT3
for(i in c("pvalue", "log2FoldChange")){
print(sum(duplicated(df_s.vs.c.noNa.m[, i])))
print(sum(is.na(df_s.vs.c.noNa.m[, i])))
print(sum(df_s.vs.c.noNa.m[, i] == 0))
}
## [1] 4
## [1] 0
## [1] 5
## [1] 0
## [1] 0
## [1] 0
There are 2 duplicated p-values (we dont mind about duplicated log2FC). There are 2 values with p=0. Examine values.
df_y.vs.c.noNa.m[duplicated(df_y.vs.c.noNa.m$pvalue) | duplicated(df_y.vs.c.noNa.m$pvalue,
fromLast = T), ]
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | padj <dbl> | stat <dbl> | ||
|---|---|---|---|---|---|---|---|---|
| 1840 | C1qb | 1.473839 | 0.1023997 | 0.37116599 | 0.2659839 | 0.4268813 | 1.112359 | |
| 2457 | Chd3 | 1595.129092 | -3.2379115 | 0.07534639 | 0.0000000 | 0.0000000 | -43.033316 | |
| 3442 | Dpep2 | 1.473839 | 0.1023997 | 0.37116599 | 0.2659839 | 0.4268813 | 1.112359 | |
| 11154 | Pgrmc1 | 1244.715326 | -3.7129484 | 0.09596356 | 0.0000000 | 0.0000000 | -38.778987 |
#STAT3
df_s.vs.c.noNa.m[duplicated(df_s.vs.c.noNa.m$pvalue) | duplicated(df_s.vs.c.noNa.m$pvalue,
fromLast = T), ]
symbol <chr> | baseMean <dbl> | log2FoldChange <dbl> | lfcSE <dbl> | pvalue <dbl> | padj <dbl> | stat <dbl> | lfcSE_not_shrunk <dbl> | ||
|---|---|---|---|---|---|---|---|---|---|
| 1241 | Armcx4 | 2485.031 | -2.431835 | 0.05679003 | 0 | 0 | -42.87583 | 0.05677956 | |
| 2140 | Cd34 | 8841.095 | -3.881954 | 0.06978926 | 0 | 0 | -55.67907 | 0.06976226 | |
| 2400 | Chd3 | 1595.129 | -5.531728 | 0.10678816 | 0 | 0 | -51.81234 | 0.10678829 | |
| 3121 | Ddx3y | 2610.027 | -3.271166 | 0.06843422 | 0 | 0 | -47.84761 | 0.06842538 | |
| 13584 | Stat3 | 19857.060 | 2.146440 | 0.04342214 | 0 | 0 | 49.49358 | 0.04340101 |
We have to add a value, to avoid that 0 becomes Inf after log transformation.
df_y.vs.c.noNa.m$pvalue[which(df_y.vs.c.noNa.m$pvalue == 0)[1]] <- 2.225074e-308
df_y.vs.c.noNa.m$pvalue[which(df_y.vs.c.noNa.m$pvalue == 0)] <- 2.225084e-308
which(df_y.vs.c.noNa.m$pvalue == 0)
## integer(0)
#STAT3
df_s.vs.c.noNa.m$pvalue[which(df_s.vs.c.noNa.m$pvalue == 0)[1]] <- 2.225074e-308
df_s.vs.c.noNa.m$pvalue[which(df_s.vs.c.noNa.m$pvalue == 0)] <- 2.225084e-308
which(df_s.vs.c.noNa.m$pvalue == 0)
## integer(0)
We changed pvalues from 0 to small values.
If you have more p-values == 0, then use “stat” column from DESeq2 output, instead of p-value.
Make ranked gene list.
v_namedRnk <- -log10(df_y.vs.c.noNa.m$pvalue) * sign(df_y.vs.c.noNa.m$log2FoldChange)
#Stat3
v_namedRnk_s <- -log10(df_s.vs.c.noNa.m$pvalue) * sign(df_s.vs.c.noNa.m$log2FoldChange)
name vector by Entrez ID
names(v_namedRnk) <- df_y.vs.c.noNa.m$ENTREZID
#STAT3
names(v_namedRnk_s) <- df_s.vs.c.noNa.m$ENTREZID
sort decreasing
v_namedRnk <- sort(v_namedRnk, decreasing = T)
#STAT
v_namedRnk_s <- sort(v_namedRnk_s, decreasing = T)
gseaKEGG <- gseKEGG(geneList = v_namedRnk, # ordered named vector
organism = "mmu",
minGSSize = 120, # minimum gene set size (# genes in set) - change to test more sets or recover sets with fewer # genes
pvalueCutoff = 0.05, # padj cutoff value
verbose = FALSE)
## Extract the GSEA results
gseaKEGG_results <- gseaKEGG@result
df_gseaKsig <- gseaKEGG_results[gseaKEGG_results$p.adjust < 0.05, ]
dim(df_gseaKsig)
## [1] 1 11
df_gseaKsig
ID <chr> | Description <chr> | setSize <int> | ||
|---|---|---|---|---|
| mmu05171 | mmu05171 | Coronavirus disease - COVID-19 - Mus musculus (house mouse) | 180 |
#STAT3
gseaKEGG_s <- gseKEGG(geneList = v_namedRnk_s, # ordered named vector
organism = "mmu",
minGSSize = 120, # minimum gene set size (# genes in set) - change to test more sets or recover sets with fewer # genes
pvalueCutoff = 0.05, # padj cutoff value
verbose = FALSE)
## Extract the GSEA results
gseaKEGG_results_s <- gseaKEGG_s@result
df_gseaKsig_s <- gseaKEGG_results_s[gseaKEGG_results_s$p.adjust < 0.05, ]
dim(df_gseaKsig_s)
## [1] 0 8
df_gseaKsig_s
ID <chr> | Description <chr> | setSize <int> | enrichmentScore <dbl> | NES <dbl> | pvalue <dbl> | p.adjust <dbl> | qvalue <dbl> |
|---|
Only one, not very useful, gensets significant.
Write GSEA results to file
write.csv(gseaKEGG_results, "gsea.y.vs.c.kegg.csv", quote=F)
gseaHm <- GSEA(geneList = v_namedRnk,
TERM2GENE = df_gsToGene)
gseaHm <- setReadable(gseaHm, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
df_gsea.h.y.c <- data.frame(gseaHm)
dim(df_gsea.h.y.c)
## [1] 3 11
df_gsea.h.y.c
ID <chr> | ||
|---|---|---|
| HALLMARK_INTERFERON_ALPHA_RESPONSE | HALLMARK_INTERFERON_ALPHA_RESPONSE | |
| HALLMARK_TNFA_SIGNALING_VIA_NFKB | HALLMARK_TNFA_SIGNALING_VIA_NFKB | |
| HALLMARK_INTERFERON_GAMMA_RESPONSE | HALLMARK_INTERFERON_GAMMA_RESPONSE |
#STAT3
gseaHm_s <- GSEA(geneList = v_namedRnk_s,
TERM2GENE = df_gsToGene)
gseaHm_s <- setReadable(gseaHm_s, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
df_gsea.h.y.c_s <- data.frame(gseaHm)
dim(df_gsea.h.y.c_s)
## [1] 3 11
df_gsea.h.y.c_s
ID <chr> | ||
|---|---|---|
| HALLMARK_INTERFERON_ALPHA_RESPONSE | HALLMARK_INTERFERON_ALPHA_RESPONSE | |
| HALLMARK_TNFA_SIGNALING_VIA_NFKB | HALLMARK_TNFA_SIGNALING_VIA_NFKB | |
| HALLMARK_INTERFERON_GAMMA_RESPONSE | HALLMARK_INTERFERON_GAMMA_RESPONSE |
3 gensets significant
gseaHm2 <- pairwise_termsim(gseaHm)
#stat3
gseaHm2_s <- pairwise_termsim(gseaHm_s)
Barplot
barplot(gseaHm, showCategory = 2)
## Error in barplot.default(gseaHm, showCategory = 2): 'height' must be a vector or a matrix
#stat3
barplot(gseaHm_s, showCategory = 2)
## Error in barplot.default(gseaHm_s, showCategory = 2): 'height' must be a vector or a matrix
dotplot
dotplot(gseaHm, showCategory=3) +
ggtitle("dotplot for ORA") +
theme(axis.text.y = element_text(size = 7))
#Stat3
dotplot(gseaHm_s, showCategory=3) +
ggtitle("dotplot for ORA") +
theme(axis.text.y = element_text(size = 7))
heatmap
heatplot(gseaHm, foldChange=v_nam, showCategory=3) +
theme(axis.text.x = element_text(size = 3.75))
#stat3
heatplot(gseaHm_s, foldChange=v_nam, showCategory=3) +
theme(axis.text.x = element_text(size = 3.75))
treeplot
treeplot(gseaHm2, hclust_method = "average")
## Error in stats::cutree(hc, nCluster): elements of 'k' must be between 1 and 3
cnetplot
cnetplot(gseaHm2, categorySize = "pvalue", foldChange = v_nam,
showCategory=3, cex_label_gene=0.5, cex_label_category = 0.5)
#STAT3
cnetplot(gseaHm2_s, categorySize = "pvalue", foldChange = v_nam_s,
showCategory=3, cex_label_gene=0.5, cex_label_category = 0.5)
In addition, you can make a gseaplot, showing the rank of the genes of the genset in the ranked gene list from the diff.exp.analysis.
gseaplot(gseaHm, geneSetID = 'HALLMARK_INTERFERON_ALPHA_RESPONSE')
The above methods don’t take into account how genes interact with each other (e.g. activation, inhibition, phosphorylation, direct/indirect interaction, ubiquitination, etc). Pathway topology methods this extra information in computing pathway-level statistics.
We will use a tool called SPIA. Here are some links to nice description of this tool: link1, link2, link3
as an input, you need a vector with sig. genes, named with entrez id
v_nam.entrez <- df_y.vs.c.noNa.sig$log2FoldChange
names(v_nam.entrez) <- df_y.vs.c.noNa.sig$ENTREZID
remove na and duplicated values
v_nam.entrez <- v_nam.entrez[!is.na(names(v_nam.entrez))]
v_nam.entrez <- v_nam.entrez[!duplicated(names(v_nam.entrez))]
as a background we use all genes tested in diff. exp. an
background_genes <- df_y.vs.c.noNa.m$ENTREZID
background_genes <- background_genes[!is.na(background_genes)]
background_genes <- background_genes[!duplicated(background_genes)]
SPIA uses KEGG Pathways Dont run it - it takes very long on the server!!!!
# spia_result <- spia(de=v_nam.entrez,
# all=background_genes,
# organism="mmu",
# plots = TRUE)
If plot is TRUE in the last command, a pdf file with Perturbation plots (SPIAPerturbationPlots.pdf) will be saved in the current working directory.
Load results from inpDir
# saveRDS(spia_result, paste(inpDir, "spia_results.rds", sep = "/"))
spia_result <- readRDS(paste(inpDir, "spia_results.rds", sep = "/"))
head(spia_result, n=20)
Name <chr> | ID <chr> | pSize <int> | NDE <int> | pNDE <dbl> | tA <dbl> | ||
|---|---|---|---|---|---|---|---|
| 1 | Cytokine-cytokine receptor interaction | 04060 | 125 | 27 | 7.634504e-10 | 9.874791 | |
| 2 | Jak-STAT signaling pathway | 04630 | 95 | 20 | 1.879019e-07 | -4.073919 | |
| 3 | Neuroactive ligand-receptor interaction | 04080 | 71 | 17 | 2.196410e-07 | 0.000000 | |
| 4 | Transcriptional misregulation in cancer | 05202 | 133 | 19 | 1.283151e-04 | -2.660223 | |
| 5 | Influenza A | 05164 | 126 | 17 | 5.634642e-04 | 21.704201 | |
| 6 | Asthma | 05310 | 13 | 5 | 4.509771e-04 | 0.000000 | |
| 7 | Autoimmune thyroid disease | 05320 | 20 | 7 | 6.298438e-05 | 0.000000 | |
| 8 | Leukocyte transendothelial migration | 04670 | 82 | 9 | 3.665249e-02 | 46.829991 | |
| 9 | Allograft rejection | 05330 | 21 | 7 | 8.999028e-05 | 0.000000 | |
| 10 | Graft-versus-host disease | 05332 | 22 | 7 | 1.257236e-04 | 0.000000 |
SPIA outputs a table showing significantly dysregulated pathways based on over-representation and signaling perturbations accumulation. The table shows the following information:
sig. pathways:
spia_result[spia_result$pGFdr < 0.05, ]
Name <chr> | ID <chr> | pSize <int> | NDE <int> | pNDE <dbl> | tA <dbl> | ||
|---|---|---|---|---|---|---|---|
| 1 | Cytokine-cytokine receptor interaction | 04060 | 125 | 27 | 7.634504e-10 | 9.874791 | |
| 2 | Jak-STAT signaling pathway | 04630 | 95 | 20 | 1.879019e-07 | -4.073919 | |
| 3 | Neuroactive ligand-receptor interaction | 04080 | 71 | 17 | 2.196410e-07 | 0.000000 | |
| 4 | Transcriptional misregulation in cancer | 05202 | 133 | 19 | 1.283151e-04 | -2.660223 | |
| 5 | Influenza A | 05164 | 126 | 17 | 5.634642e-04 | 21.704201 | |
| 6 | Asthma | 05310 | 13 | 5 | 4.509771e-04 | 0.000000 | |
| 7 | Autoimmune thyroid disease | 05320 | 20 | 7 | 6.298438e-05 | 0.000000 | |
| 8 | Leukocyte transendothelial migration | 04670 | 82 | 9 | 3.665249e-02 | 46.829991 | |
| 9 | Allograft rejection | 05330 | 21 | 7 | 8.999028e-05 | 0.000000 | |
| 10 | Graft-versus-host disease | 05332 | 22 | 7 | 1.257236e-04 | 0.000000 |
A summary Plot of the results can be obtained using the command below. The genesets above the blue and red curve are significant based on the combined p-value obtained by combining pNDE and pPERT.
plotP(spia_result, threshold=0.05)
## Error in if (sum(oky) > 0) {: missing value where TRUE/FALSE needed
Genemania is a Webtool linkToGenemania hat has stored interactions of different types/from different resources, between genes. We can use it to explore functional relationships between our sig. reg. genes, and possible relationships to other genes. Before you start using Genemania, please read the help linkToHelp or one of these tutorials tutorial1, tutorial2.
Gene symbols can be used as input. We will therefore write out our sig. genes (gene Symbols) ordered by p-value. We then can use the top e.g. 50 genes for the analysis in Genemania.
write.xlsx(df_y.vs.c.noNa.sig[order(df_y.vs.c.noNa.sig$pvalue), ][, "symbol", drop = FALSE],
"df_y.vs.c.noNa.sig.symbOnly.xlsx")
# copy to inpDir
file.copy("df_y.vs.c.noNa.sig.symbOnly.xlsx", inpDir)
## [1] FALSE
STRING linkToString is a Webtool that has stored interactions of different types/from different resources, between genes. We can use it to explore functional relationships between our sig. reg. genes, and possible relationships to other genes.
These are some helpful resources how to work with STRING: linkToTutorial1, linkToPublication.
In addition to identify networks between provided genes, it can also perform ORA and GSEA analysis.
write.xlsx(l_df.res.all$y.vs.c.noNa.sigPadjFcUp,
file = "y.vs.c.noNa.sigPadjFcUp.xlsx")
# copy to inpDir
file.copy("y.vs.c.noNa.sigPadjFcUp.xlsx", inpDir)
## [1] FALSE
open xlsx file, and copy Gene symbols into “multiple protein” search box. Then press Search (organism is detected automatically). Press Continue to confirm correct ID identification.
Ignore the Large Network warning, and press continue.
In the legend tab, you see the inputted genes, with a short
description of their functions.
In the settings tab, you can modify different settings. You can switch
the network type, the meaning of network edges, and which interaction
sources are considered.
Furthermore, you can set the minimum required interaction score.
At the analysis tab, you see the results of the enrichment analysis. You can color the nodes by the Pathways they are in, or you can select a single gene, and check in which genset it is.
fwrite(data.frame(entrezId = names(v_nam.entrez),
rank = v_nam.entrez),
file = "twoColEntrezRankForString.csv")
v_namedRnk
## 20848 20397 17063 74016 12984
## 2.900643e+02 1.799662e+02 1.787236e+02 1.710772e+02 1.659187e+02
## 383787 171207 59126 76108 18007
## 1.604836e+02 1.542307e+02 1.516464e+02 1.492305e+02 1.380007e+02
## 18715 227937 239217 227620 14687
## 1.343826e+02 1.331897e+02 1.214323e+02 1.212545e+02 1.198017e+02
## 18712 18439 20527 12048 20910
## 1.168415e+02 1.117257e+02 1.111728e+02 1.084804e+02 1.032206e+02
## 14727 244418 20908 19122 104445
## 1.024602e+02 1.020278e+02 9.973689e+01 9.589364e+01 9.446703e+01
## 70599 12983 14664 18286 72349
## 9.203889e+01 9.063597e+01 8.933688e+01 8.800249e+01 8.746978e+01
## 228775 170750 231147 14127 170718
## 8.716182e+01 8.633005e+01 8.629724e+01 8.555330e+01 8.513510e+01
## 320706 20515 18024 20893 20322
## 8.487650e+01 8.480825e+01 8.441473e+01 8.412078e+01 8.389796e+01
## 384009 228875 23872 56454 69601
## 8.363232e+01 8.249228e+01 8.192964e+01 8.009802e+01 7.959839e+01
## 71648 59043 16909 83768 227695
## 7.876267e+01 7.767889e+01 7.742366e+01 7.474339e+01 7.377179e+01
## 65973 19261 230784 319520 14683
## 7.375109e+01 7.337548e+01 7.333213e+01 7.214022e+01 7.193435e+01
## 99237 100678 72630 19177 56325
## 7.163053e+01 7.118235e+01 7.113865e+01 7.076159e+01 7.069012e+01
## 108115 70433 13836 66865 14824
## 6.962518e+01 6.952887e+01 6.870699e+01 6.844477e+01 6.756922e+01
## 56738 18550 73192 14086 228836
## 6.735634e+01 6.723339e+01 6.722348e+01 6.628765e+01 6.561700e+01
## 56791 77975 68713 19248 74411
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## 67615 19087 22591 73451 68021
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## -1.848481e+01 -1.854493e+01 -1.854880e+01 -1.856258e+01 -1.856447e+01
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## -1.865426e+01 -1.867446e+01 -1.869469e+01 -1.872053e+01 -1.872100e+01
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## 19280 18574 66880 18685 20364
## -1.903760e+01 -1.904572e+01 -1.910023e+01 -1.914681e+01 -1.917924e+01
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## -1.922832e+01 -1.923433e+01 -1.927748e+01 -1.929468e+01 -1.943584e+01
## 14661 14042 218639 66923 50927
## -1.946432e+01 -1.946514e+01 -1.948839e+01 -1.957962e+01 -1.963072e+01
## 26365 233913 242466 12606 30878
## -1.975592e+01 -1.977187e+01 -1.981596e+01 -1.984991e+01 -1.988301e+01
## 12314 15950 226407 140742 210376
## -1.992045e+01 -1.995005e+01 -1.998610e+01 -2.004739e+01 -2.007942e+01
## 216350 72194 225876 14007 219249
## -2.013423e+01 -2.026166e+01 -2.040641e+01 -2.040728e+01 -2.043180e+01
## 11370 11532 328329 71795 16506
## -2.054519e+01 -2.065655e+01 -2.066751e+01 -2.067884e+01 -2.080227e+01
## 110197 21753 246177 68977 21402
## -2.081885e+01 -2.082343e+01 -2.089660e+01 -2.099222e+01 -2.104045e+01
## 29819 54354 67771 17688 19241
## -2.108422e+01 -2.118178e+01 -2.123755e+01 -2.130314e+01 -2.131678e+01
## 66912 64291 19889 110611 211922
## -2.134413e+01 -2.145314e+01 -2.146131e+01 -2.149964e+01 -2.152514e+01
## 11758 12366 330959 50932 19091
## -2.158824e+01 -2.171028e+01 -2.183609e+01 -2.184980e+01 -2.187004e+01
## 13136 13655 12380 242687 20308
## -2.198389e+01 -2.201806e+01 -2.202091e+01 -2.204133e+01 -2.205948e+01
## 67949 226652 230863 12847 109151
## -2.208233e+01 -2.209670e+01 -2.211590e+01 -2.219280e+01 -2.226579e+01
## 20444 18477 76843 56619 17347
## -2.232633e+01 -2.242786e+01 -2.243003e+01 -2.250136e+01 -2.257649e+01
## 634731 12499 57265 67102 320404
## -2.259847e+01 -2.259915e+01 -2.262391e+01 -2.262639e+01 -2.272856e+01
## 329154 434341 65079 232334 13211
## -2.282490e+01 -2.284115e+01 -2.287024e+01 -2.296826e+01 -2.300086e+01
## 226861 320982 59287 16765 98193
## -2.300771e+01 -2.302881e+01 -2.315084e+01 -2.315643e+01 -2.315913e+01
## 19192 21391 216080 66385 16592
## -2.322330e+01 -2.322875e+01 -2.323937e+01 -2.332596e+01 -2.340527e+01
## 216543 68839 11566 210293 18193
## -2.351684e+01 -2.362566e+01 -2.367942e+01 -2.372100e+01 -2.375293e+01
## 27371 381290 59090 12469 76123
## -2.385463e+01 -2.386226e+01 -2.387822e+01 -2.395388e+01 -2.400113e+01
## 52463 67707 66505 58246 68616
## -2.412388e+01 -2.427703e+01 -2.433314e+01 -2.435580e+01 -2.438863e+01
## 16565 75974 66151 74116 109032
## -2.456665e+01 -2.458458e+01 -2.470273e+01 -2.471517e+01 -2.483064e+01
## 14701 101994 328232 192176 77976
## -2.494384e+01 -2.503396e+01 -2.522001e+01 -2.524921e+01 -2.531907e+01
## 230761 21885 18637 320586 215690
## -2.532055e+01 -2.542386e+01 -2.558271e+01 -2.578931e+01 -2.596623e+01
## 207278 226562 232288 102098 67664
## -2.596658e+01 -2.605682e+01 -2.606733e+01 -2.607622e+01 -2.633259e+01
## 74081 12774 227333 16870 17159
## -2.639080e+01 -2.644380e+01 -2.662501e+01 -2.662923e+01 -2.664267e+01
## 20365 15441 13713 16192 19062
## -2.666812e+01 -2.680573e+01 -2.683988e+01 -2.687570e+01 -2.690871e+01
## 14026 74464 12925 195727 12798
## -2.707306e+01 -2.709763e+01 -2.721152e+01 -2.724725e+01 -2.736126e+01
## 73469 233405 16019 213539 20230
## -2.745295e+01 -2.746790e+01 -2.751809e+01 -2.761785e+01 -2.761792e+01
## 214597 66514 193385 269224 19073
## -2.772229e+01 -2.780737e+01 -2.792900e+01 -2.798563e+01 -2.809123e+01
## 236511 72042 64214 70160 18032
## -2.828440e+01 -2.830905e+01 -2.836098e+01 -2.838189e+01 -2.857485e+01
## 19253 15381 70186 170753 20661
## -2.886372e+01 -2.897296e+01 -2.905180e+01 -2.909125e+01 -2.911469e+01
## 76709 394432 245595 192119 13032
## -2.912533e+01 -2.920517e+01 -2.933037e+01 -2.952980e+01 -2.955863e+01
## 16541 17215 16579 18566 13714
## -2.967891e+01 -2.970448e+01 -2.972509e+01 -2.973313e+01 -2.973655e+01
## 68944 14964 68514 18106 19125
## -2.981447e+01 -2.982794e+01 -2.984584e+01 -2.986352e+01 -3.010941e+01
## 21346 268445 17434 24064 108829
## -3.014037e+01 -3.022344e+01 -3.028257e+01 -3.028525e+01 -3.034041e+01
## 328795 69035 11545 226841 381694
## -3.034309e+01 -3.042580e+01 -3.056458e+01 -3.059438e+01 -3.065353e+01
## 22355 20649 93840 14131 18203
## -3.073458e+01 -3.089179e+01 -3.108135e+01 -3.108374e+01 -3.111849e+01
## 22778 67460 15488 66824 73341
## -3.119150e+01 -3.133982e+01 -3.158023e+01 -3.161124e+01 -3.164245e+01
## 80898 382045 12479 229473 14645
## -3.204381e+01 -3.212607e+01 -3.218028e+01 -3.221080e+01 -3.221508e+01
## 22341 20363 140580 11774 100038725
## -3.238451e+01 -3.267617e+01 -3.277156e+01 -3.291925e+01 -3.306979e+01
## 74513 108147 102636203 67869 16398
## -3.307213e+01 -3.308863e+01 -3.333354e+01 -3.334727e+01 -3.345675e+01
## 216197 20658 52440 19663 78334
## -3.361876e+01 -3.364743e+01 -3.374171e+01 -3.408345e+01 -3.413617e+01
## 103841 226432 72465 140577 100039888
## -3.420663e+01 -3.468042e+01 -3.518511e+01 -3.522517e+01 -3.537463e+01
## 319670 230316 19651 74106 17993
## -3.577501e+01 -3.581375e+01 -3.603205e+01 -3.647545e+01 -3.651021e+01
## 76088 101056162 170737 259302 93966
## -3.659563e+01 -3.691719e+01 -3.741105e+01 -3.749811e+01 -3.791699e+01
## 330286 70945 77065 16576 52882
## -3.807434e+01 -3.828043e+01 -3.913264e+01 -3.917426e+01 -3.926784e+01
## 229905 55927 20849 14194 103677
## -3.938657e+01 -4.017073e+01 -4.018758e+01 -4.026193e+01 -4.048411e+01
## 22719 226830 269233 22083 14972
## -4.053104e+01 -4.071213e+01 -4.090484e+01 -4.107216e+01 -4.124494e+01
## 19395 71787 17219 66043 667799
## -4.140105e+01 -4.179203e+01 -4.192427e+01 -4.201288e+01 -4.209862e+01
## 74185 15002 545030 93736 59054
## -4.217274e+01 -4.233988e+01 -4.239183e+01 -4.241247e+01 -4.244234e+01
## 17260 108672 20588 215243 72462
## -4.303193e+01 -4.308185e+01 -4.326826e+01 -4.341543e+01 -4.392390e+01
## 22344 66892 219140 18019 108686
## -4.418905e+01 -4.445410e+01 -4.452748e+01 -4.466339e+01 -4.477381e+01
## 22350 218693 72433 17685 71330
## -4.570632e+01 -4.593650e+01 -4.689054e+01 -4.740028e+01 -4.839698e+01
## 13171 225207 27494 56229 67792
## -4.913579e+01 -4.935576e+01 -4.978452e+01 -4.992187e+01 -5.003554e+01
## 53623 217946 56878 668218 16451
## -5.035323e+01 -5.074829e+01 -5.093589e+01 -5.155879e+01 -5.161069e+01
## 20452 76041 19265 20742 66725
## -5.227720e+01 -5.229611e+01 -5.258472e+01 -5.265596e+01 -5.267994e+01
## 16432 12986 66307 70155 19649
## -5.287157e+01 -5.322065e+01 -5.335488e+01 -5.393285e+01 -5.486249e+01
## 56526 71592 76707 76429 14744
## -5.524985e+01 -5.538930e+01 -5.553155e+01 -5.595173e+01 -5.601732e+01
## 64213 233328 223697 226594 78798
## -5.648619e+01 -5.708142e+01 -5.732178e+01 -5.743436e+01 -5.788853e+01
## 14525 26914 268973 240752 73690
## -5.812591e+01 -5.828190e+01 -5.884724e+01 -5.984129e+01 -6.057751e+01
## 12557 12183 22596 83925 26432
## -6.079037e+01 -6.139320e+01 -6.517781e+01 -6.631423e+01 -6.720011e+01
## 70729 67392 83669 18201 108116
## -6.744012e+01 -6.848802e+01 -6.963742e+01 -7.025400e+01 -7.044124e+01
## 27412 107684 106522 170748 14025
## -7.193323e+01 -7.319603e+01 -7.326184e+01 -7.494057e+01 -7.595600e+01
## 56792 218629 171170 50766 236915
## -7.777856e+01 -8.095908e+01 -8.134638e+01 -8.210289e+01 -8.324326e+01
## 27357 108052 17304 12523 54204
## -8.419715e+01 -8.645548e+01 -8.740168e+01 -8.938799e+01 -9.459737e+01
## 13007 240754 70549 11732 50934
## -9.546244e+01 -9.615622e+01 -9.667404e+01 -9.819803e+01 -1.030445e+02
## 76438 17085 244202 56228 16985
## -1.095454e+02 -1.109358e+02 -1.131828e+02 -1.149241e+02 -1.156694e+02
## 12506 18583 13641 76132 12490
## -1.176728e+02 -1.240534e+02 -1.290506e+02 -1.330333e+02 -1.332771e+02
## 72198 252838 11600 665574 330450
## -1.358195e+02 -1.377297e+02 -1.392665e+02 -1.573439e+02 -1.641227e+02
## 75547 107895 104099 53328 216848
## -1.695769e+02 -1.707981e+02 -2.127129e+02 -3.076527e+02 -3.076527e+02
You can also do GSEA with String. Strings needs a 2 column Table,
first column with IDs, second with value that rank genes. We can use the
ranked vector named with Entrez IDs generated before. We write it out,
and then upload the file to String. Select “Proteins with Values/Ranks”
in the Search Menu, and upload the table. Confirm mapping of IDs.
You will get the sig. enriched Terms from different databases. You can
see the rank of the genes in the enriched terms in the ranked list by
selecting the specific term.
From ORAs and GSEA select results (Y640F.vs.dsRed and STAT3.vs.dsRed) from one database (the same, or different - ideally the one with a appropriate number of pathways, or the one where you can best interpret the results).
In presentation, put ORAs from different Databases of STAT3-wt.vs.dsRed and Y640F.vs.dsRed next to each other, and shortly describe what you see (dont mind if your interpretation is correct or not - it is sometimes hard to say, without additional exploration of results).
Alternatively, you can use compareCluster to directly compare both analysis in one plot.